This invention relates generally to methods of treating vascular diseases.
Atherosclerosis and abdominal aortic aneurysm (AAA) are inflammatory diseases that involve extensive extracellular matrix degradation and vascular wall remodeling. Cardiovascular events correlate with the presence of inflammation, and atheroma tend to rupture at sites of matrix remodeling (Davies et al., 94 Circulation 213-22 (1996); Ghorpade et al., 800 Ann. N.Y. Acad. Sci. 138-50 (1996)).
However, the mechanisms of degradation of the arterial extracellular matrix in these various contexts remain unclear. In particular, cysteine proteases have previously received little consideration in this mechanism, even though macrophages and smooth muscle cells with greatly expanded lysosomal compartments figure prominently in the histopathology of atherosclerotic plaques. Several cysteine endoproteases (cathepsin K, cathepsin L, and cathepsin S, have potent elastolytic activity. Although elastolytic and collagenolytic cysteine proteases may participate in vascular wall remodeling, cathepsin K has only been observed in bone, and only one study has demonstrated cathepsin K mRNA and protein expression in human breast carcinoma (Littlewood-Evans et al., 57 Cancer Res. 5386-390 (1997)).
Although cathepsins are thought to generally reside in and function optimally within acidic lysosomes, cathepsins also function extracellularly at or near the cell surface. Monocyte-derived human macrophages become markedly elastolytic during in vitro culture and predominantly use cysteine proteases to degrade extracellular elastin. In the presence of pro-inflammatory cytokines found in atheroma, cultured smooth muscle cells secrete active cathepsin S capable of degrading extracellular elastin (Sukhova et al., 102 J. Clin. Invest. 576-83 (1998)). Changes in the concentration of cathepsins B and C localized to aortic aneurysm have recently been described (Gacko et al., 50(2) Pol. J. Pathol. 83-86 (1999)), as has increased expression of cathepsin B in the advancing edge of colorectal tumors (Hirai et al., 30(6) Hum. Pathol. 680-86 (1999)).
By regulating protease activities, protease inhibitors also play a pivotal role in tissue remodeling (see, Werb et al., 857 Ann. N.Y. Acad. Sci. 110-18 (1998)). The most abundant extracellular inhibitor of cysteine proteases is cystatin C (see, Barrett et al., 120 Biochem. Biophys. Res. Commun. 631-36 (1984)). Cystatin C is expressed in virtually all organs of the body, and is present in high concentration in biological fluids. However, aside from a rare mutation in cystatin C that leads to its precipitation as amyloid in cerebral blood vessels and causes cerebral hemorrhage, no evidence has thus far implicated cystatin C in disease.
While cystatin C preparations have been described for the treatment of viral w disease (U.S. Pat. No. 5,432,264), protection of the nervous system from papain (Hiltke et al., 78(8) J. Dent. Res. 1401-409 (1999)), diagnosis of amyloidosis (U.S. Pat. No. 5,270,165), and identification of therapeutic agents to prevent neuronal cell death (U.S. Pat. No. 5,866,318), the role of cystatin C in vascular disease remains unclear. Until now, it has been unknown whether cystatin C expression actually changes in situ in diseases in which inflammation is prominent, and how changes in cystatin C levels might be effected. Thus, there remains a need in the art for the elucidation of the role cystatin C plays in vascular disease, including atherosclerosis, and for the development of therapeutics to prevent and treat vascular elastic laminae breakdown.
The invention is directed to methods of preventing and treating vascular diseases, inflammatory disorders, and vascular tumors by inhibiting cysteine proteases. Specifically, the invention is directed to the inhibition of two cysteine proteases active at sites of vascular injury, cathepsin S and cathepsin K, by cystatin C, but also includes inhibition of other elastolytic proteases, such as cathepsin L.
The pathogenesis of atherosclerosis and abdominal aortic aneurysm involves elastic laminae breakdown. Elastolytic cysteine proteases are overexpressed at sites of arterial elastin damage, but whether endogenous local inhibitors counterbalance these proteases has previously been unknown. While the cysteine protease inhibitor cystatin C is normally expressed in vascular wall smooth muscle cells, cystatin C is severely reduced in both atherosclerotic and aneurysmal aortic lesions. Increased abdominal aortic diameter correlates inversely with serum cystatin C levels. In vitro, cytokine-stimulated vascular smooth muscle cells secrete elastolytic cathepsin S and cathepsin K. The elastolytic activity of these enzymes is blocked by as little as 10 ng/ml of recombinant cystatin C or by transforming growth factor-xcex21 (TGF-xcex21)-induced cystatin C secretion.
The invention provides methods of preventing the development of and treating vascular disease in a subject by administering an inhibitor of a cysteine protease. In one aspect the subject has not been diagnosed with a vascular disease. Examples of vascular diseases include athersclerosis or aneurismal aortic lesions. Cysteine proteases include cystatins, e.g. cystatin C polypeptide.
In another aspect the invention provides methods of diagnosing a subject at risk of or with vascular disease. A subject at risk is a subject who for example has a family history of vascular disease or has been diagnosed as having a predisposition to vascular disease (e.g., familial hypercholesterolemia or hereditary angiodema). In addition a subject at risk also may be an individual who demonstrates behavioral or environmental risk factors associated with vascular disease. Such factors include for example, gender, advancing age, cigarette smoking, high blood pressure, diabetes, obesity, lack of physical activity, abnormal blood cholesterol homocysteine levels or decreased serum cystatin C levels as compared to a normal healthy adult value. The more risk factors a person has, the greater the likelihood of developing vascular disease. Several of these risk factors are interrelated. Obesity, lack of exercise, and cigarette smoking can raise blood pressure and adversely influence blood cholesterol levels.